All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
A. Allergic Inflammation
Allergic inflammation is a complex phenomenon, involving various cell types such as inflammatory and structural cells. Mast cells are the well-established initiators of allergic inflammation, attracting, activating and finally interacting with other inflammatory cells, mainly the eosinophils. Allergic inflammation comprises a variety of pathologies, such as asthma, allergic rhinitis, allergic conjunctivitis, atopic eczema etc. Among these diseases, and as an example, asthma is the most common illness of early childhood, counting for up to 20% in Western countries and currently increasing [Busse WW, Lemanske RF Jr. (2001) N Engl J Med. 344:3501].
Experimentation in the field of allergy has provided insights into the cellular and molecular mechanisms underlying these pathologies. These investigations have led to the understanding that the allergic response is often biphasic. The first, early phase is initiated by mast cell activation (see below), while the second, late phase is brought about by the infiltration of inflammatory cells, predominantly T-cells, eosinophils and some basophils [Broide D H, Firestein G S. (1991) J Clin Invest. 88:10482]. Knowledge, however, has not yet yielded efficacious therapeutic means. Currently used approaches offer either symptomatic relief (i.e. anti-histamines and anti-leukotrienes) or a non-selective anti-inflammatory treatment (i.e. glucocorticosteroids). In addition, newly developed immunopharmacological treatments targeting a single antibody (e.g. IgE), T cell cytokine (e.g. anti-IL-5) or several transcription factors (e.g. STAT-6, GATA-3 or FOG-1) have not proven efficient as yet.
B. Mast Cells
Mast cells are tissue dwelling, FcεRI bearing cells containing prominent cytoplasmic granules. Besides having a pivotal role in allergic reactions, they are also involved in fibrosis, tumors, autoimmune diseases and innate immunity. Mast cells are widely distributed throughout the body, in connective tissues and on mucosal surfaces where they are usually located in close proximity to blood vessels and peripheral nerves. Therefore, they are exposed to environmental stimuli such as microorganisms and allergens with which they can react, both within minutes and/or over a period of hours, and undergo regulated secretion of preformed and newly synthesized mediators.
Upon activation, mast cells release a variety of inflammatory mediators including pre-formed granule constituents (e.g. histamine, proteoglycans and proteases), PGD2, LTC4, PAF, and to a lesser extent, LTB4, and a variety of cytokines (e.g. IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-13, RANTES, IFN-γ, TGF-β, TNF-α, and GM-CSF) [Puxeddu I. et al. (2003) Int J Biochem Cell Biol. 35:16013].
In addition to the classical “allergic” IgE-dependent mast cell activation that is triggered by the binding of allergens to two adjacent IgE molecules bound to FcεRI, there are other ways of mast cell stimulation. IgE-independent mast cell activation may be particularly important in the setting of the late phase and in chronic inflammation. Notably, while anti-IgE therapy is now approved for the treatment of asthma, it only induces a modest improvement. This highlights the involvement of non-IgE dependent pathways in the development of asthma as well as the need for new targets for therapeutic intervention. Indeed, work done in the inventors' laboratory (as well as in others) has shown that numerous mediators are capable of activating mast cells [Piliponsky A. M. et al. (2003) Blood 101:1898-4; Feldweg, A. M. et al. (2003) Eur. J. Immunol. 33:2262-8]. Among them, stem cell factor (SCF), which is critically responsible for mast cell differentiation, survival, proliferation, maturation, chemotaxis, adhesion, as well as activation, and Nerve Growth Factor (NGF) which also induces mast cell activation.
The IgE-independent stimulation of mast cells can also be triggered by polybasic compounds that share similar structural features essential for their activity such as compound 48/80, neuropeptides (VIP, CGRP, substance P, neurotensin), and eosinophil derived-major basic protein (MBP) [Piliponsky A. M. et al. (2003) id ibid.].
Eosinophils are bone marrow-derived granulocytes that differentiate under the regulation of the transcription factors GATA-1&2, and c/EBP, and the cytokines IL-3, GM-CSF and IL-5 (“eosinophil survival cytokines”) [Kaatz Maa et al (2004) Int. J. Mol. Med. 14:1055-160]. Notably, CD4+Th2 cells are the main producers of these cytokines [Umland SP et al (1998) Am J Respir Cell Mol Biol 18:631-42]. Eosinophils normally enter the blood and migrate into the gastrointestinal tract, but in inflammatory states they can accumulate in various tissues. Here they may survive for several days due to effects of the locally released “survival cytokines,” before programmed cell death occurs. Eosinophils are associated with host defense mechanisms in parasitic infestations and are implicated in the pathogenesis of allergic, immunological and malignant disorders as well as a variety of idiopathic hypereosinophilic syndromes [Bain, B. J. (2004) Am J Hematol 77:82-5; Klion, A. D. et al (2004) J. Allergy Clin. Immunol. 113:30-7]. In LAR (late asthmatic response), eosinophils may be responsible for tissue damage (mostly epithelial) through the release of their cytotoxic granule proteins. In addition, evidence is emerging implicating eosinophils as effector cells involved in the tissue repair and fibrosis associated with asthma [Levi-Schaffer, F. et al. (1999) Proc Natl Acad Sci USA 96:9660-5].
Eosinophils store preformed granule mediators, like major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO); synthesize lipid mediators, like PAF, LTC4, and PGE2, as well as proinflammatory and immunoregulatory cytokines and chemokines, like IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-13, IL-16, GM-CSF, SCF, NGF, TNF-α, TGF-β, INF-γ, MIP-1, RANTES and eotaxin [Piliponsky, A. M. et al. (2002) Mol Immunol 38:1369]. The eosinophil basic proteins were found to be highly toxic in vitro to respiratory epithelial cells, at concentrations detected in biological fluid from patients with asthma. Furthermore, eosinophils produce matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1/2. These cells also contain heparanase and are a source for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and b-fibroblast growth factor (b-FGF) [Munitz, A. et al. (2004) Allergy 59:268-75], clearly indicating their role in asthma-associated and fibrosis with asthma.
Activation of eosinophils and consequent mediator release, both in allergic setting and in other diseases can be induced by a series of agonists. In fact, receptors for several pro-inflammatory mediators (i.e. C5a, PAF), cytokines (i.e. IL-5, GM-CSF, IL-3, IL-2, IFNγ etc.), immunoglobulins (i.e. IgG, IgA) and chemokines (i.e. CCR3) [Munitz (2004) id ibid.] are expressed on the eosinophil's surface. However, the role of these receptors in promoting eosinophil activation in vivo (especially in the setting of chronic allergic airway inflammation) is not known. This is not just an academic question, since blockade of eosinophil activation is currently being pursued for the treatment of asthma. Recent results with anti-IL-5 therapy have reaffirmed the need to identify the fundamental mechanisms of eosinophil activation, since this reagent did not have a significant impact on eosinophil degranulation in asthmatics [Kay, A. B. et al (2003) Am J Respir Crit Care Med 167:1586-7]. Eosinophils have also been found to express several additional inhibitory/activatory Ig-superfamily cell surface receptors also expressed in mast cells, such as LIR-3/ILT-5, LIR-1/ILT-2, LIR-2/ILT-4, LIR-7/ILT-1 [Tedla N. et al. (2003) Proc. Natl. Acad. Sci. USA 100:1174-9] and siglecs [Nutku, E. et al (2003) Blood 101:5014-20].
Eosinophils encounter mast cells in the tissue during the late phase of the allergic inflammatory process. Recently, evidence has emerged indicating that there is an important cross-talk between these two cells. Work done in the inventors' laboratory has shown that eosinophil survival is enhanced by mast cell-derived TNF-α via TNF-αRI and TNF-αRII [Temkin, V. et al. (2001) Cytokine 15:20-6]. Furthermore, the preformed mast cell-derived tryptase induces IL-6 and IL-8 production and release from human peripheral blood eosinophils by PAR-II initiating the mitogen-activated protein kinase (MAPK)/AP-1 pathway, while GM-CSF produced by IgE-activated mast cells induces eosinophil survival and eosinophil cationic protein (ECP) release. Human lung-derived mast cells become responsive to MBP when co-cultured with fibroblasts, by a process dependent on membrane-bound SCF. Notably, eosinophils also synthesize SCF and NGF. Altogether, all this strengthens the importance of mast cells and eosinophils in the late and chronic stages of allergic inflammation [Temkin V. et al. (2002) J Immunol. 169:2662; Hartman M. et al. (2001) Blood 97:10865-6; Solomon, A. et al. (1998) J. Allergy Clin. Immunol. 102:454-60].
It has recently become clear that mast cell degranulation is regulated by additional surface activatory and inhibitory receptors such as FcγRIIB, gp49A/B1/B2, PIR-B, LIRs/ILTs and sialic acid binding Ig-like lectins (siglecs) that are expressed on mast cells and functional on murine and human mast cells [Katz H R. (2002) Curr Opin Immunol. 14:6987].
C. Inhibitory Receptors
It has become increasingly apparent that both mast cells and eosinophils express several inhibitory receptors belonging either to the Ig receptor superfamily (characterized by a single V-type Ig-like domain in the extracellular portion such as KIRs, LIRs/ILTs, LAIR, gp49B1, etc.) or to the c-type (calcium dependent) lectin superfamily (such as MAFA, CD94/NKG2A). This large family of immune inhibitory receptors can be identified by a consensus amino acid sequence, the immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is present in the cytoplasmic domain of these molecules. The archetype ITIM sequence is composed of 6 amino acids (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val), where X denotes any amino acid. Upon activation, these inhibitory receptors undergo tyrosine phosphorylation, often by a Src family kinase, which provides a docking site for the recruitment of cytoplasmic phosphatases having a Src homology 2 (SH2) domain such as SHP-1,-2 and SHIP-1,-2 [Ravetch J V, Lanier L L. (2000) Science. 290:848].
As previously described, mast cells can be activated by IgE-dependent (FcεRI mediated) or -independent stimuli. Activation of mast cells via IgE-dependent mechanisms results in rapid recruitment of syk and lyn to tyrosine phosphorylated residues in the intracellular component of the FcεRI receptor termed ITAM (immunoreceptor tyrosine-based activatory motif). The consequence of this action is histamine and other preformed mediators release and synthesis, and release of lipid mediators by a rapid process that is completed in less than 30 minutes. In addition, SCF and NGF, which activate mast cells, are dependant on Src family kinases. Interestingly, both IgE-dependent and independent stimuli are regulated by inhibitory receptors at least in vivo in mice models. Thus, recruitment of SHP-1, -2 and SHIP-1,-2 that dephosphorylate ITAM domains or kinase activity result in downregulation of mast cell activation. This inhibition has been thoroughly described for the gp49B1 inhibitory receptor on murine mast cells, where co-ligation of the inhibitory receptor with FcεRI resulted in inhibition of secretory granule mediator (histamine, β-hexosaminidase) and LTC4 release [Katz H. R. et al. (1996) Proc Natl Acad Sci USA. 93:10809].
IRp60 (inhibitory receptor protein 60) is an inhibitory receptor belonging to the Ig superfamily. It is expressed on many cell types such as T-cells, NK cells and granulocytes. Cross-linking of IRp60 on NK cells, results in down-regulation of NK cytolyitic activity. In addition, treatment of IRp60 with sodium pervanadate led to marked IRp60 tyrosine phosphorylation and association with both SHP-1 and SHP-2 [Cantoni C. et al. (1999) Eur J Immunol. 29:3148]. Furthermore, IRp60 cross-linking inhibited the cytolitic activity of T-cell clones in re-directed killing assays using anti-CD3 mAb. Importantly, the ligand of IRp60 is yet unknown.
D. Bi-Specific Antibodies (BsAb).
In recent years, antibody therapy has become a new treatment modality for a vast array of diseases such as cancer, malaria and asthma. Nonetheless, it is widely agreed that the efficacy of antibodies requires further improvement.
Bi-specific antibodies are proteins that have two different binding specificities, usually designed to recognize two different antigens on different cells. Thus, one binding site is specific for an antigen on the target cell (i.e. infected or cancer cell) while the other binding site recognizes specifically an antigen on the immune effector cell. Accordingly, the effector-cell mechanisms will be exerted upon the target cell leading to an appropriate immune response [Hudson, P. J. et al. (2003) Nat. Med. 9:129-34.
First-generation bi-specific antibodies were produced by fusing two established hybridoma cell lines to form quadromas [Milstein C, Cuello A C. (1983) Nature. 305:537] or by chemical cross-linking of respective F(ab′) fragments [Karpovsky B. et al. (1984) J Exp Med. 160:1686]. In vitro, in vivo and clinical studies done with such bi-specific antibodies confirmed the therapeutic potential of such a treatment [van de Winkel J. G. et al. (1997) Immunol Today. 18:562].
A novel approach with bi-specific antibodies has been to have the two different antigens to be recognized present in the same cell. Daeron et al. [US 2004/0038894] describe the possibility that a bi-specific antibody which would recognize an inhibitory KIR—Killer cell Immunoglobulin Receptors, which are mostly expressed in NK cells and function as cell surface receptors for MHC Class I molecules—and simultaneously a stimulatory receptor, e.g. ITAM-bearing receptors such as an activating Ig receptor, FcεRI, CD3/TCR, to cite just a few, would have the ability to cross-link said stimulatory receptor with said KIR, intra- or extra-cellularly. Said cross-linking would then result in the regulation of the activation of said stimulatory receptor, to which the ultimate outcome would be the modulation of immune and inflammatory responses. This prediction is actually confirmed by Tam et al. [Tam, S. W. et al. (2004) Allergy 59(7):772], who described the generation of a bi-specific antibody against human IgE and human FcεRII. Said antibody was able to inhibit antigen-induced histamine release by human mast cells and basophils.
The present inventors have characterized the expression of inhibitory receptors in mast cells and eosinophils, and particularly the expression of the inhibitory receptor IRp60 in these cells (see Examples below).
In the present invention, the inventors describe the generation of bi-specific antibodies that are able to bind and activate the inhibitory receptor IRp60 in a cell-specific manner, due to its target-cell specific module.
The particular focus of the present study is to target cells involved in the allergic response, like mast cells, eosinophils and basophils, and therefore provide a new, more efficient, cell specific agent for the treatment of allergy-related illnesses.
Thus, it is an object of the present invention to provide a BsAb which recognizes and activates the inhibitory receptor IRp60 (first component of the BsAb) and one other marker (second component of the BsAb) specific for mast cells, eosinophils or basophils, said marker being an activator (or a receptor) whose signal transduction pathway is inhibited by the activation of the inhibitory receptor (i.e., the first component of the BsAb).
Other uses and objects of the invention will become clear as the description proceeds.